Cross-linked collagen comprising metallic anticancer agents

ABSTRACT

The disclosure describes collagen constructs comprising anticancer agents, preferably, platinum, and related methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/815,834, filed on Mar. 15, 2013 which claims priority toU.S. provisional patent application No. 61/728,198, filed on Nov. 19,2012. The content of the prior applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to cross-linked collagen constructs comprisingmetal containing anticancer agents or metallic anticancer alkylators,preferably alkylators comprising platinum, and related methods.

BACKGROUND OF THE INVENTION

Koob et al. have described methods of producing nordihydroguaiareticacid (NDGA) polymerized collagen fibers for various biomedicalapplications, some with tensile strengths similar to that of naturaltendon (e.g., about 91 MPa). See, for example, Koob and Hernandez,Material properties of polymerized NDGA-collagen composite fibers:development of biologically based tendon constructs, Biomaterials 2002January; 23 (1): 203-12; and U.S. Pat. No. 6,565,960, the contents ofwhich are hereby incorporated by reference as if recited in full herein.

SUMMARY OF THE INVENTION

This invention is directed to cross-linked collagen constructs,preferably which are biocompatible, comprising a releasablemetal-containing anticancer agent incorporated thereto. The constructscan be used for in vivo delivery and, in some embodiments, theanticancer agent is incorporated into the construct so that there is asustained release of the anticancer agent.

In one aspect of this invention, the biocompatible cross-linked collagenis provided as a construct with a releasable anticancer agent and,preferably, a platinum anticancer agent. Such constructs provide thebeneficial properties of the cross-linked collagen as well as impartanticancer properties to the construct.

In another aspect, the biocompatible cross-linked collagen constructprovides a sustained release of the anticancer agent including animmediate release, an intermediate release and an extended release. Incertain embodiments, the anticancer agent is continuously released fromthe collagen construct.

In one aspect, provided herein is an anticancer cross-linked collagenconstruct comprising:

a cross-linked collagen comprising collagen and one or morecross-linking agents and

an anticancer amount of platinum incorporated into the construct atleast in part through the cross-linking agent.

As used herein, “incorporated” or “bound” refers to the metal beingcovalently and/or non-covalently, such as via cation-pi interactions,attached to the construct, and includes multiple covalent bonds orchelates between the metal atom and the construct.

In one embodiment, the cross-linking agent comprises a 1,2-benzoquinoneand/or a 1,2-dihydroxy phenyl moiety. In another embodiment, theplatinum is chelated at least in part to the benzoquinone and/or the1,2-dihydroxyphenyl moiety. In another embodiment, the cross-linkingagent is selected from the group consisting of nordihydroguaiaretic acid(NDGA), 3,4-dyhydroxyphenylalanine, dopamine, 3,4-dihydroxybenzaldehyde,3,4-dihydroxybenzoic acid, a carbodiimide, glutaraldehyde or another di-or multi aldehyde, formaldehyde, tannic acid, isocyanates, pluronics,and epoxy resins. In another embodiment, the cross-linking agent isNDGA. In another embodiment, the platinum comprises platinum (II) and/orplatinum (IV). In another embodiment, the platinum comprisescis-(NH₃)₂Pt(II),

wherein

denotes binding to the cross-linked collagen construct. In anotherembodiment, the platinum incorporated into the construct is present inan amount of between about 0.1% to about 30%.

In another embodiment, the anticancer amount of platinum is released invivo in contact with an aqueous medium. In another embodiment, theanticancer cross-linked collagen construct provides a sustained releaseof platinum, wherein the sustained release comprises a plurality ofrelease rates including an immediate release, an intermediate release,an extended release or any combination of release rates thereof. Inanother embodiment, an effective amount of platinum is released fromabout 1 minute to about 60 days, or any range therein.

As used herein, an “anticancer amount of platinum” refers to an amountof platinum incorporated into the anticancer cross-linked collagenconstruct of this invention that when contacted in vitro or in vivo withcancer cells inhibit or kill cancer cells or tissue, and inhibits orkills, preferably, 50%, more preferably, 90%, and still more preferably99% of such cells or tissue. As used herein, “effective amount ofplatinum” refers to an amount of platinum that is, preferably, releasedfrom an anticancer cross-linked collagen construct of this invention andthat is sufficient to inhibit or kill, in vitro or in vivo, preferably,50%, more preferably, 90%, and still more preferably 99% of cancer cellsor tissue. An effective amount of platinum can improve one or morecancer symptoms, and/or ameliorate one or more cancer-side effects,and/or prevent and/or impede invasiveness and/or metastasis of cancer.In certain embodiments, an anticancer amount of platinum can differ froman effective amount of platinum.

As used herein “cancer amenable to treatment with platinum alkylators”refer to those cancers that are treated with one or more platinumalkylators, non limiting examples of which include cisplatin,carboplatin, oxaliplatin, satraplatin, as are well known to the skilledartisan. Non limiting examples of such cancers include non-small celllung, breast, ovarian, testicular, and bladder cancer. “Alkylators” or“alkylating agents” as used herein are agents that can alkylate, ortransfer an electrophilic metal moiety, such as a platinum moiety to,nucleic acids and/or proteins as is well known to the skilled artisan.

In one embodiment, the invention is directed to a construct comprising across-linked collagen construct and an anticancer amount of platinumincorporated into the construct to provide an anticancer, cross-linkedcollagen construct. In certain aspects, the cross-linked collagenconstruct is cross-linked with one or more cross-linking agent selectedfrom the group consisting of nordihydroguaiaretic acid (NDGA), crosslinkers including 2-9 1,2-dihydrophenyl moieties,3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxybenzaldehyde,3,4-dihydroxybenzoic acid, a carbodiimide, glutaraldehyde and anotherdialdehyde and a multialdehyde, formaldehyde, tannic acid, isocyanatessuch as di and multi-isocyanates, alpha-diazo pyruvates such as di andmulti-alpha-diazo pyruvates, pluronics, preferably such as L61, L121,F68, F108, and epoxy resins. In exemplary embodiments, the cross-linkedcollagen construct is at least cross-linked with NDGA.

In yet another embodiment of the invention, the platinum incorporatedinto the construct is bound to a quinone group and/or a catechol grouppresent in the cross-linked collagen construct. In other embodiments ofthe invention, the platinum incorporated into the construct may be boundto a basic nitrogen atom, non limiting examples of which include amino,or mono- or di-alkylated amino, and imidazole. In certain aspects, theplatinum is platinum (+2) and/or platinum (+4). In certain preferredaspects, the platinum anticancer agent comprises a cis diamino platinummoiety. As used herein the amino refers to ammonia or a cycloalkylamine, or a heteroaryl or a heterocyclyl amine, where the platinumcoordinating nitrogen atom can be a part of the ring system orderivatize the ring system. Examples of cis diamino platinum moietiesand other platinum moieties useful in this invention are described,e.g., in Kostova, Recent Patents on Anti-Cancer Drug Discovery, 2006, 1,1-22, which is incorporated herein in its entirety by reference. Inother aspects, the platinum incorporated into the construct is presentin an amount of between about 0.1% to about 30%.

In additional aspects of the invention, the anticancer amount ofplatinum is released in vivo, e.g., during degradation of the construct.In certain aspects, the anticancer, cross-linked collagen constructprovides a sustained release of platinum, wherein the sustained releasecomprises a plurality of release rates including an immediate release,an intermediate release, an extended release or any combination ofrelease rates thereof. In other aspects, the plurality of release ratesare adjusted to provide a suitable range of release rates, wherein therange of release rates comprises from about 1 minute to about 60 days,or any range therein.

The anticancer collagen constructs can be formulated variously dependingon their mode of delivery and their delivery site. In certain preferredembodiments, the anticancer collagen constructs are powdered ormicronized. In some embodiments, the powdered or micronized constructsare compacted into a shape such as a pellet or an implant shaped as thegraphite tube in a pencil. Unit lengths or doses of such shaped soliddosage forms are also provided. Such solid forms of the anticancerconstructs can be administered topically to a site needing anticancertreatment, or may be administered into a patient by using dry, solidinjection techniques well known and/or commercially available, or theirobvious modifications. Non-limiting examples of such solid injectionstechniques include, the Glide SDI, solid injection system.

In other embodiments, the anticancer collagen constructs or theirpowdered or micronized or other forms are formulated as a viscous fluid.Such viscous formulations may preferably include non-aqueous organicliquids, which can include polymers such as polyethylene glycols,Polaxamer® polymers, and the like, and/or small molecule organicsolvents. Such viscous formulations may be administered, preferably sitespecifically, using high pressure syringes that are well known and/orcommercially available, or obvious modifications thereof. Non limitingexamples of such high pressure syringes include those described in U.S.Pat. No. 6,503,244 (incorporated herein by reference) and the likes.

In another aspect, provided herein is a method of manufacturing ananticancer cross-linked collagen construct comprising cross-linkedcollagen and an anticancer amount of platinum, wherein the platinum isincorporated into the construct at least in part through thecross-linking agent, the method comprising providing the cross-linkedcollagen comprising collagen and one or more cross-linking agents; andcontacting the cross-linked collagen with a cis-diaqua or a cis-dihaloplatinum complex, to provide the anticancer cross-linked collagenconstruct.

As used herein, a cis-diaqua platinum complex contains 2 water moleculesbound to platinum in cis or adjacent configuration, and a cis-dihaloplatinum complex contains 2 halogen, preferably chloride groups, boundto platinum in cis or adjacent configuration.

Another embodiment of the invention is directed to a method ofmanufacturing a construct comprising: providing a cross-linked collagen;and contacting the cross-linked collagen construct with an anitcanceramount of platinum to chemically bind the platinum to the cross-linkedcollagen construct to provide a therapeutically effective amount ofanticancer platinum in the construct, thereby producing an anticancercross-linked collagen construct.

In another aspect, provided herein is a method of treating a subjectsuffering from cancer amenable to treatment with platinum alkylators,the method comprising administering an anticancer cross-linked collagenconstruct in the subject, wherein the construct comprises (a)cross-linked collagen comprising collagen and one or more cross-linkingagents and (b) platinum incorporated therein at least in part throughthe cross-linking agent, to administer an effective amount of platinuminto the subject.

In yet another embodiment, the invention is directed to a method oftreating a subject in need of treatment for a cancer treatable with aplatinum anticancer agent, comprising: a) implanting a medical constructin a subject, wherein the medical construct comprises cross-linkedcollagen and an anticancer amount of platinum incorporated therein toprovide a therapeutically effective amount of anticancer platinum in theconstruct, and b) releasing the anticancer agent from the construct in aplurality of in vivo release rates, thereby inhibiting cancer. In oneembodiment the effective amount of platinum is released from theconstruct in a plurality of in vivo release rates. Methods ofdetermining the therapeutically effective amount, appropriate mode ofadministration of the compounds and compositions provided herein will beapparent to the skilled artisan upon reading this disclosure and basedon other methods known to them.

It is noted that aspects of the invention described with respect to oneembodiment, may be incorporated in a different embodiment although notspecifically described relative thereto. That is, all embodiments and/orfeatures of any embodiment can be combined in any way and/orcombination. These and other objects and/or aspects of the presentinvention are explained in detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of operations that can be used to carry outembodiments of the present invention. Preferably, the collagen constructincorporating the therapeutic agent is a collagen construct comprisingplatinum as provide herein.

FIG. 2 is a digital photograph of exemplary NDGA-collagen tubes (10)according to embodiments of the present invention of varying diameterand length.

DETAILED DESCRIPTION

The present invention is described more fully hereinafter with referenceto the accompanying drawings, in which embodiments of the invention areshown. This invention may, however, be embodied in many different formsand should not be construed as limited to the embodiments set forthherein; rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinvention to those skilled in the art. Before describing this invention,the following terms are defined.

Definition

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Collagen construct,” as used herein, refers to a device and/or materialthat comprises biocompatible cross-linked collagen. The collagenconstruct can be in finished or final form for use or in an unfinishedor pre-final form. The collagen constructs of the present invention cancomprise natural collagen, natural collagenous tissue, syntheticcollagen, and/or any combination thereof “Synthetic collagen” as usedherein, refers to collagen material that has been formed and/orchemically and/or physically created or altered from itsnaturally-occurring state. As such, synthetic collagen may include, butis not limited to, collagen material in the form of a gel, gelatin,fibril, slurry, hydrogel or a film, each of which is discussed infurther detail herein below.

The term “cross-linked collagen construct” or “biocompatiblecross-linked collagen construct” refers to collagen cross-linked with across-linking agent that is preferably biocompatible and capable ofbinding and releasing a metallic anti-cancer agent. In one embodiment,the cross-linking agent contains two or more functionalities which arereactive with collagen as well as one or more functionalities which arecapable of binding and releasing the metallic anti-cancer agent.“Anticancer cross-linked collagen construct” refers to a cross linkedcollagen construct further comprising an anticancer agent, preferably, ametallic anti-cancer agent, more preferably, platinum.

In one embodiment, the cross-linking agent contains the same ordifferent functionalities which can both cross-link collagen andreversibly bind the anticancer agent. Preferably, the cross-linkingagents are catechol containing cross-linking reagents including, by wayof example, NDGA as well as:

where L is a covalent bond or a linking group of from 1 to 10 atomswhich comprise 1-8 carbon atoms and 0 to 4 heteroatoms selected from thegroup consisting of oxygen, sulfur, NH, and the like.

Such catechol containing cross-linking reagents permit a variety oflengths between the collagen fibrils as well as binding to differentfunctionalities. In one exemplary embodiment, the catechol cross-linkingreagent is NDGA, which is well known in the art.

“Cross linkers including 2-9 1,2-dihydroxyphenyl moieties” refer to, inpreferred embodiments, compounds of formula:

In some embodiments, the phenyl ring containing one or more hydroxygroups can further contain alkylthio (alkyl-S—) and/or amino, or in someembodiments, the hydroxy groups in the phenyl ring can be replaced byalkylthio (alkyl-S—), cyano, and/or amino, groups. In some embodiments,the phenyl ring or the hydroxy substituted phenyl ring can be replacedby a heteroaryl ring containing one or more nitrogen atoms, preferably(—N═) nitrogen atom(s), that can bind the metal. Such other substitutedphenyl or heteroaryl compounds are made according to methods well knownto a skilled artisan.

In some embodiments, the chelators are bis amino acids where the twoamino acid moieties are covalently bonded by a linker such as L. Theamino acid moieties are suitable modified so as to bind to collagen, aswill be well know to a skilled artisan. For example, and withoutlimitation, aspartic acid, and/or glutamic acid can be useful as the twoamino acids.

By including a variety of ligands that bind the anti-cancer metal,preferably platinum, strongly to weakly, the release rate of that metalcan be controlled.

Such other cross-linkers will be apparent to the skilled artisan uponreading this disclosure.

A “carbodiimide” refers to a compound of formula X—N═C≡N—X, wherein eachX independently is C₁-C₆ alkyl optionally substituted with 1-2dialkylamino groups, or is C₅-C₆ cycloalkyl.

“C_(m)” when placed before a group refers to that group containing mcarbon atom(s).

“Alkyl” refers to a hydrocarbyl radical, preferably monovalent,containing 1-12 carbon atoms. Non limiting examples of alkyl includesmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, andthe like.

“Cycloalkyl” refers to a cyclic hydrocarbyl radical, preferably,monovalent, containing 3-10 carbon atoms and includes bicyclic radicals.Non limiting examples of cycloalkyl include cycloproyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like.

“Dialdehyde” refers to a compound of formula OHC-L-CHO, wherein L isdefined as above. “Multialdehyde” refers to a compound of formula:

wherein, each L independently is defined as above, p is 1-9, and X isdefined as follows. X can be a moiety containing one or more,preferably, 1-9, 1,2-dihydroxyphenyl moiety. Or, X can be nitrogen, isan hydrocarbyl moiety optionally containing 1-10 heteroatoms, or is aglycosidic moiety or an amino acid and such other multifunctional moietyto which the L groups are bound. Dialdehydes and multialdehydes are wellknown to the skilled artisan.

“Di-isocyanate” refers to a compound of formula ONC-L-CNO, wherein L isdefined as above. “Multi-isocyanate” refers to a compound of formula:

wherein X, L, and p are defined as above. Di-isocyanates andmulti-isocyanates are well known to the skilled artisan.

“Di-alpha-diazo pyruvate” refers to a compound of formulaN₂C(O)C(O)-L-C(O)C(O)N₂, and “multi-alpha-diazo pyruvate” refers to acompound of formula:

wherein X, L, and p are defined as above. Di-alpha-diazo pyruvates andmulti-alpha-diazo pyruvates are well known to the skilled artisan.

“Pluoronics” refers to block copolymers of formula:HO(C₂H₄O)_(a)(CH₂CH(CH₃)O)_(b)(C₂H₄O)_(a)H where such polymers andvalues of a and b are well known to the skilled artisan.

Exemplary biocompatible cross-linked collagen constructs (which aresometimes referred to herein as “collagen constructs” or merely“constructs”), include, but are not limited to, patches, such as woundbed patches, muscle or organ patches, cardiac patches, hernia patches,skin patches, burn treatment patches, and skin/tissue repair patches;cuffs; blood vessel (artery, vein, and the like) repair material; valvereplacements or valve repair material; auto-graft material; allo-graftmaterial; xenograft material; nerve guides; tubes; tendon sleeves, suchas sleeves that can reside about repairing tendon to prevent or inhibitadhesions; indwelling tubes for delivery of anticancer agents; ducts,such as lymphatic, hepatic, pancreatic and cystic ducts; tubes, such asureter and urethra tubes; collagen fiber; collagen gel; sutures; cords;twisted cords; ligament and/or tendon prosthesis; cables; braids;ribbons; staples; rivets; sponges; and the like. Further examples anddescription of devices are described in U.S. Pat. No. 7,901,455; U.S.Patent Application Publication Nos. 2008/0161917, 2008/0188933,2008/0200992, 2009/0216233, 2009/0287308, 2010/0094318, and2010/0094404; U.S. patent application Ser. Nos. 13/153,665 and13/105,353; and U.S. Provisional Patent Application No. 61/450,179,which are incorporated herein by reference.

The collagen constructs of the present invention can be dry or partiallyhydrated. The term “dry” as used herein means the construct has amoisture content of less than about 5% by weight of the construct. Theterm “partially hydrated” as used herein means that the construct has amoisture content that is less than about 50%, typically less than about75% of the moisture content at full hydration, measured ex vivo after 24hours in a saline bath at ambient conditions. Thus, the construct canhave a moisture content of less than about 25% by weight of theconstruct, such as less than about 15% by weight of the construct. Incertain embodiments, the construct comprises at least one drymanufactured collagen fiber.

In some embodiments of the present invention, the collagen constructcomprises at least one collagen fiber. A collagen fiber can be anelongate continuous length of fiber formed of denatured (gelatin) and/ornon-denatured collagen (e.g., whole or fragmented native collagen fibersfrom tendon, skin, or other sources). An elongate collagen fiber canhave a length of at least about 0.25 inches (0.63 cm), typically greaterthan about 0.5 inches (1.27 cm), such as between about 1-30 inches (2.54cm to 76.2 cm) or between about 1 m to about 100 m. In some embodimentsof the present invention, a collagen construct comprises a plurality ofelongate NDGA cross-linked collagen fibers. In certain embodiments ofthe present invention, a collagen construct comprises at least onepolymerized elongate collagen fiber.

Examples of fiber configurations include a single fiber, a plurality offibers, a fiber bundle, or a plurality of fiber bundles and/or fiberstwisted, woven or braided that define a twisted, woven or braided fiberbundle and/or fiber construct.

The term “patch” refers to a piece or segment of biomaterial that can beplaced on and/or affixed to target anatomical structure, typically softtissue, to treat, protect, repair and/or reinforce a target site. Thepatch can be any geometric shape but is typically substantially planarand may, in position, conform to the shape of underlying or overlyingtissue.

The term “implantable” and derivatives thereof means the device can beinserted, embedded, grafted or otherwise acutely or chronically attachedor placed in or on a subject.

The terms “winding” and “wound” and derivatives thereof means to wrapabout an object or center at least once, typically repeatedly, e.g., toturn in a series of circular motions. In some embodiments, at least onecollagen fiber (multiple fibers, one or more fiber bundles) turns orrotates its circumferential position about a centerline or long axis.The winding may define a coil (e.g., a series of connected typicallysubstantially concentric rings or spirals), woven and/or braided fiberarrangement with a number of revolutions or turns about a core and/ortube, typically in a regular pattern (but an irregular pattern may alsobe used) about a length of at least one layer of a tube or cylindricalshape.

The present invention finds use in medical applications and animalstudies. The term “medical” includes both human and veterinary uses.Suitable subjects of the present invention include, but are not limitedto avians and mammals.

In particular embodiments, the subject is “in need of” the methods ofthe present invention, e.g., the subject may benefit from a surgicalprocedure implanting a collagen construct of the present invention, suchas a prosthesis or other device. In certain embodiments, afterimplantation, the collagen constructs of the present invention canconfer a therapeutic and/or prophylactic effect to the subject, such asprevent a disease and/or clinical symptom, reduce the severity of adisease and/or clinical symptom relative to what would occur in theabsence of the methods of the prevent invention, and/or delay the onsetand/or progression of a disease and/or clinical symptom. The methods ofthe present invention can provide complete and/or partial treatmentand/or protection. In particular embodiments, after implantation in oradministration to a subject, the collagen constructs of the presentinvention treat and/or inhibit and/or protect against cancer,preferably, those that are treated with platinum anticancer agents, inthe subject.

I. Anticancer Agents

Embodiments of the present invention are directed to biocompatiblecross-linked collagen constructs with chemically bound anticancer agentsfor in vivo delivery at different release rates over time for medicaluse.

The collagen constructs of the present invention, in particularembodiments, can provide a therapeutically effective amount of ananticancer agent. “Therapeutically effective amount,” as used herein,refers to an amount of an anticancer agent that elicits atherapeutically useful response in treating an existing medicalcondition and/or preventing or delaying the onset of a medical conditionfrom occurring in a subject. In particular embodiments, the collagenconstruct provides a therapeutically effective amount of an anticanceragent for at least about 5 days, such as at least about 15 days, atleast about 20 days, at least about 30 days, at least about 60 days, atleast about 100 days, or more. In other embodiments, the collagenconstruct provides a therapeutically effective amount of an anticanceragent for the lifetime of the collagen construct. As used herein, theterm “lifetime of the collagen construct” is the period of timebeginning substantially when the collagen construct is implanted andextending until the time the collagen construct is removed or degrades,breaks down, delaminates, denatures, resorbs, absorbs, decomposes, ordisintegrates, such that the collagen construct no longer serves itsstructural and/or functional purpose (i.e., the useful lifetime). Insome embodiments of the present invention, an anticancer agent isreleased before, during, after, or upon degradation of the construct, orany combination thereof.

In some embodiments, the anticancer agent comprises an anticancer heavymetal cation. In certain embodiments, the anticancer agent is platinum.The term “platinum,” as used herein, includes all platinum salts suchas, platinum (+2), and or (+4) salts, preferably, platinum (+2) salts,still more preferably cis-diamino platinum salts. Accordingly, it isunderstood that all forms of anticancer platinum salts are contemplatedto be suitable to provide a therapeutically effective dose of anticancerplatinum according to the collagen constructs of the present invention.

The anticancer agent can present in a collagen construct of the presentinvention in an amount of between about 0.1% to about 30%, such asbetween about 0.1% to about 10%, about 1% to about 5%, about 1% to about30%, about 3% to about 25%, or about 5% to about 15% by weight of thecollagen construct. In some particular embodiments of the presentinvention, the anticancer agent is present in an amount of about 1% toabout 5% by weight of the collagen construct.

The platinum can be present in the collagen construct in an amountbetween about 10,000 μg to about 300,000 μg, on average, or any rangetherein, such as about 10,000 μg to about 200,000 μg, about 15,000 μg toabout 80,000 μg, about 20,000 μg to about 50,000 μg, or about 50,000 μgto about 150,000 μg per gram of the collagen construct, on average. Inparticular embodiments of the present invention, the heavy metal ispresent in an amount of about 15,000 μg to about 50,000 μg per gram ofthe collagen construct, on average.

The amount of the anticancer agent present in a collagen construct canbe based on the weight of the collagen construct. In particularembodiments, the amount of the anticancer agent present in a collagenconstruct can be based on the dry weight of the collagen construct(i.e., having a moisture content of less than about 5% by weight of theconstruct). In other embodiments, the amount of the anticancer agentpresent in a collagen construct can be based on the weight of thecollagen construct having a low moisture content (i.e., having amoisture content of less than about 25% by weight of the construct, suchas less than about 15% by weight of the construct).

The anticancer agent can be incorporated into and/or onto the collagenconstructs of the present invention. “Incorporate” and grammaticalvariants thereof, as used herein, refer to an anticancer agent beingpresent in the collagen constructs of the present invention. The term“incorporate” and grammatical variants thereof, is intended to includeembodiments where the anticancer agent is present on one or more of thesurfaces of the collagen construct and/or present in one or more layersof the collagen construct. “Incorporate” is intended to includeembodiments where an anticancer agent is bound to the collagenconstruct, such as through specific and/or nonspecific types of binding.Exemplary types of chemical bonds through which the anticancer agent canbind to the collagen construct include, but are not limited to, covalentbonds, noncovalent bonds, ionic bonds, metallic bonds, or anycombination thereof. In some embodiments of the present invention, theanticancer agent can bind to and/or complex with the collagen and/orother materials or compounds in the collagen constructs of the presentinvention. In certain embodiments, the anticancer agent nonspecificallybinds to the collagen construct to provide an immediate bolus release ofthe anticancer agent from the collagen construct and specifically bindsto the collagen construct to provide a sustained release of theanticancer agent from the collagen construct. In other embodiments, therelease of the anticancer agent from the collagen construct can be dueto a combination of specific and nonspecific binding of the anticanceragent to the collagen construct.

In particular embodiments of the present invention, the anticancer agentcan bind to and/or complex with a moiety or group present in thecollagen construct. In some embodiments, the anticancer agent can bindto and/or complex with a moiety or group present in cross-linkedcollagen, e.g., NDGA cross-linked collagen, in the collagen constructsof the present invention. “Moiety” and “group” are used interchangeablyherein to refer to a portion of a molecule present in the collagenconstruct, typically having a particular functional and/or structuralfeature, e.g., a linking group (a portion of a molecule connecting twoother portions of the molecule). Exemplary functional groups include,but are not limited to, amino, sulfhydryl, carbonyl, hydroxyl, alkoxy,carboxyl, silyl, silyloxy, hydrocarbyl, cycloalkyl, aryl, thio,mercapto, imino, halo, cyano, nitro, azido, sulfoxy, phosphoryl, oxy,quinone, catechol, and the like. In particular embodiments, thefunctional group is a quinone and/or a catechol.

“Quinone,” as used herein refers to a compound similar to1,4-benzoquinone and derivatives thereof with one or more carbonylgroup(s) in an unsaturated ring (Scheme 1). Exemplary quinones include,but are not limited, to 1,4-benzoquinone, 1,2-benzoquinone,1,4-naphthoquinone, 9-10-anthraquinone, acetimidoquinone, alizarin,alkannin, 1-aminoanthraquinone, anthrimide, chimaphilin, chloranil,2,6-dimethoxyquinone, duroquinone, emodin, fusarubin,2-methylanthraquinone, menadione, oosporein, parvaquone, perezone,plumbagin, rhodoquinone, rufigallol, rufigallol, terreic acid,ubiquinones, aurantiogliocladin, nitranilic acid, and any combinationthereof.

“Catechol,” as used herein, refers to a compound similar to1,2-benzenediol and derivatives thereof with one or more hydroxylgroup(s) in an unsaturated ring (Scheme 2). Exemplary catechols include,but are not limited to, 1,2-benzenediol, 2,3-dihydroxynaphthalene,1,3-benzenediol, nordihydroguaiaretic acid, adrenalone, catechin,nitrocatechol, 3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid,deoxyepinephrine, dobutamine, dopamine, dopexamine, epinephrine,nordefrin, 3-pentadecylcatechol, tiron, and any combination thereof

If the collagen construct comprises a quinone group and/or a catecholgroup, the anticancer agent can bind to and/or complex with the quinonegroup and/or catechol group. Exemplary anticancer agents that can bindand/or complex with a quinone and/or a catechol group, include,preferably platinum, (including its combined forms such as salts andcomplexes with carriers), anions such as chloride, oxoanions, andhyperoxide. A catechol can oxidize to a quinone and a quinone can bereduced to a catechol. In some embodiments of the present invention,upon or during the binding of a anticancer agent, a catechol group canbe oxidized to a quinone or a quinone can be reduced to a catechol. Inparticular embodiments of the present invention, the anticancer agent isan anticancer agent, e.g., platinum, that binds to and/or complexes witha quinone and/or a catechol group present in the collagen construct. Insome embodiments, a quinone and/or catechol group is present in thecollagen construct as a result of the collagen construct beingpolymerized with a cross-linking agent, such as NDGA.

The crosslinked collagen-Pt constructs of this invention are prepared bycontacting appropriate Pt salts, preferably, Pt(II) salts with acrosslinked collagen. Preferably, the Pt salt is a cis diamino Pt(II)dichloro or, yet more preferably, a cis diamino Pt(II) diaqua salt. Anillustrative and non-limiting example is shown below:

Excess Pt salts, not bound to the crosslinked collagen, can be removedby complexed with a resin, such as Chelex. A crosslinked collagen-Pt(II)construct can be converted to the corresponding Pt(IV) construct byoxidation, for example with H₂O₂.

In some embodiments of the present invention, the collagen construct istreated with an agent to modify the collagen construct's ability toincorporate an anticancer agent. In particular embodiments, the agentmodifies or adds a functional group present in the collagen construct toincrease and/or enhance the incorporation of an anticancer agent intoand/or onto the collagen construct. Exemplary types of reactions throughwhich a modification can be made include, but are not limited to, redoxreactions, substitution reactions, addition reactions, eliminationreactions, rearrangement reactions, biochemical reactions, and anycombination thereof. In particular embodiments, a redox reaction isperformed to modify and/or change the oxidation state of a functionalgroup present in the collagen construct, e.g., a quinone group and/or acatechol group. In certain embodiments, the agent can be a polymerizingagent, such as, but not limited to, NDGA. In other embodiments, theagent blocks (e.g., partially or completely) a functional group presentin the collagen construct, which optionally can subsequently beunblocked, to allow for one or more anticancer agents and/or othermaterials to be incorporated into and/or onto the collagen construct.

In certain aspects, the greater the affinity of the anticancer agent forthe collagen construct, the more stable the coordination of thecomposition due to the ionic interaction between the anticancer agentand the collagen construct. Conversely, the lower the affinity of theanticancer agent for collagen construct, the less stable thecoordination of the composition. Thus, for applications where it isdesired to provide a slower, faster or combination of release rates ofthe anticancer agent sequestered in the collagen construct, the bindingaffinity of the anticancer agent can be modified to achieve the desiredrelease rate(s) using the methods disclosed herein.

In various embodiments, it is contemplated that at least three affinityphases correlating to release rates can be achieved: (1) a mobile phase;(2) low affinity phase; and (3) high affinity phase, wherein the phasesare arranged by order of increasing affinity of the anticancer agent forthe collagen construct. In one aspect, the mobile phase correlates tounbound and/or weakly bound anticancer agent to the collagen construct.In another aspect, the low affinity phase correlates to non-specific andspecific binding of the anticancer agent to moieties or groups presentin the collagen construct. In another aspect, the high affinity phasecorrelates to binding of the anticancer agent to quinone and/or catecholgroups present in the collagen construct.

It is to be understood that various binding affinities can be achievedusing the methods disclosed herein depending on the desired releaserate(s). In an exemplary embodiment, a plurality of release rates can beachieved by introducing an anticancer agent, for example, platinum, ontoa cross-linked collagen construct, such that the anticancer agent isincorporated into and/or onto the collagen construct via a mobile phase,low affinity and high affinity phase. In some aspects, the utilizationof different affinities of the anticancer agent will allow the skilledartisan to make a collagen construct having a desired rate of release.In these aspects, for example, the addition of an amount of anticanceragent that is less than necessary to bind higher affinity groups willprovide a slower release, without an immediate release. In anotherexample, an addition of anticancer agent in amount that is sufficient tosaturate higher affinity groups will provide a faster release than theformer example, because the anticancer agent is present in an amountsufficient to bind to additionally bind to lower affinity components,such as but not limited to, hydroxyls, amines and carboxyl groups, whichwill lead to a more immediate release.

In various embodiments, the one or more anticancer agents can elute orbe released from the collagen construct over a period of time. Theanticancer agent can elute or be released from the collagen constructcontinuously and/or substantially continuously over a period of time.“Substantially continuously,” as used herein refers to a release of ananticancer agent all or part of the time such that on average therelease of the anticancer agent still confers an overall beneficialeffect on the subject.

Thus, there may be some short, intermittent and/or regular time periodsin which an anticancer agent is not being released, but the overallbeneficial effect of the anticancer agent on the subject remains. Insome embodiments, the release rate of an anticancer agent can vary overa period of time and/or there can be multiple release rates of ananticancer agent. Alternatively, the release rate of an anticancer agentcan be substantially constant (i.e., on average varying less than about30%, 20%, 15%, 10%, or 5%) over a period of time.

In some embodiments, the release rate of a anticancer agent can besubstantially constant for a period of a time and vary over anotherconsecutive or nonconsecutive period of time and vice versa. In otherembodiments, there can be periods of time in which no anticancer agentis released. The release of an anticancer agent, in some embodiments,can occur in random and/or sequential releases of the same or varyingconcentration. When there is more than one anticancer agent present inthe collagen construct, the releases of the anticancer agents canoverlap or the release rates can occur at different times. Further, whenthere is more than one anticancer agent present in the collagenconstruct, the release rates can be the same or the release rates can bedifferent.

The collagen construct can have one or more release rates of ananticancer agent. For example, the collagen construct can have 1, 2, 3,4, 5, or more release rates of an anticancer agent from the collagenconstruct. In certain embodiments, an NDGA-collagen construct comprisingplatinum, provided according to this invention, provides for two releaserates of the platinum from the NDGA-collagen. In another embodiment, forthe first release, after incubating the NDGA-collagen in normal salineat about 37° C. for about 1 day, about 25% of the total amount ofplatinum incorporated into and/or onto the NDGA-collagen is releasedfrom the NDGA-collagen. In another embodiment, over the course of thenext 29 days, about 35% of the total amount of platinum incorporatedinto and/or onto the NDGA-collagen is released from the NDGA-collagen.Thus, in certain embodiments, after about 30 days, about 60% of thetotal amount of platinum incorporated into and/or onto the NDGA-collagenis released from the NDGA-collagen. Accordingly, about 40% of the totalamount of platinum incorporated into and/or onto the NDGA-collagenremains bound to the NDGA-collagen after about 30 days.

The amount of an anticancer agent released from the collagen constructin the one or more releases over a period of time, e.g., about 1 minuteto about 100 days.

The amount of an anticancer agent released from a collagen construct canbe measured by standard procedures, such as, but are not limited to,quantitative chemical analytical and/or bioanalytical techniques such asatomic absorption spectroscopy, mass spectrometry such as inductivelycoupled plasma mass spectrometry (ICP-MS), gas chromatography,immunoassays, and/or any combination thereof.

To measure the amount of an anticancer agent released from a collagenconstruct, the construct can be placed in a saline solution at aspecified temperature for a desired period of time. The saline solutioncan have an osmolarity of between about 100 mOsm/L to about 1000 mOsm/Lor any range therein, such as between about 100 mOsm/L to about 500mOsm/L or between about 250 mOsm/L to about 350 mOsm/L. In particularembodiments, the saline solution is normal saline. The temperature canbe between about −80° C. to about 80° C. or any range therein, such asbetween about −80° C. and about −10° C., about −20° C. to about 15° C.,about 5° C. and about 80° C., or about 30° C. and about 40° C. Inparticular embodiments, the temperature is about 37° C. The period oftime can be any duration of time in which the amount of the anticanceragent released is desired to be known, such as about 15 minutes, about 1hour, about 5 hours, about 1 day, about 5 days, about 30 days, about 100days, or more. After which time the amount of the anticancer agentreleased into the saline can be measured, as described above, andsubsequently the rate of release can be calculated. Alternatively, asample (e.g., blood or urine) can be taken from the subject in which theconstruct is implanted and the sample can be analyzed using methods suchas those described above.

To accelerate the test to determine the amount of an anticancer agentreleased from a collagen construct over a desired duration of time, suchas the lifetime of the construct, an enzyme can be added to the salinesolution. Exemplary enzymes include, but are not limited to, pepsin,bromelain, chymopapain, chymotrypsin, collagenase, ficin, papain,peptidase, proteinase A, proteinase K, trypsin, microbial proteases, andcombinations thereof. In certain embodiments of the present invention,the enzyme is a collagenase. The amount of the one or more enzyme(s)added to the saline solution can be adjusted to accelerate thedegradation of the collagen construct over a desired period of time.After the enzymatic degradation of the collagen construct for thedesired amount of time, the amount of the anticancer agent released intothe saline can be measured, as described above, and subsequently therate of release can be calculated.

In other aspects, the release of an anticancer agent from the collagenconstruct can allow for the collagen construct to retain its ability tokill and/or inhibit microorganisms (e.g., bacteria, viruses, etc.) overan extended period of time. Thus, the collagen construct can provide asustained anticancer effect.

In some embodiments of the present invention, the collagen construct hasat least two different rates of release of at least one anticancer agentfrom the collagen construct. The two different rates of release can beof the same anticancer agent or of two or even more anticancer agents.In other embodiments of the present invention, the collagen constructhas at least three different rates of release of an anticancer agentfrom the collagen construct. The three different rates of release can beof the same anticancer agent or of two or more anticancer agents.

In one aspect, the collagen construct can have an initial release of ananticancer agent from the collagen construct. The initial release of theanticancer agent can be a short-term and/or an “immediate” bolus releaseof the anticancer agent from the collagen construct after implantation.The amount of an anticancer agent released from the collagen constructin the initial release can be from about 1% to about 40% or any rangetherein, such as about 10% to about 30%, about 15% to about 35%, orabout 20% to about 30% of the total amount of an anticancer agentincorporated into and/or onto the collagen construct from about 1 minuteto about 1 day or any range therein, such as between about 15 minutes toabout 20 hours or between about 30 minutes to about 15 hours afterimplantation. In particular embodiments, about 20% to about 30% of thetotal amount of an anticancer agent incorporated into and/or onto thecollagen construct is released after about 1 day after implantation.

In another aspect, the collagen construct can have a second and/or anintermediate release of an anticancer agent from the collagen construct.The intermediate release can provide for a prolonged release, incomparison to the immediate release described above, of the anticanceragent from the collagen construct and can allow for the collagenconstruct to retain its ability to kill and/or inhibit microorganisms(e.g., bacteria, viruses, etc.) over an extended period of time. Theamount of an anticancer agent released from the collagen construct inthe second release can be from about 20% to about 100% or any rangetherein, such as about 25% to about 75% or about 30% to about 40% of thetotal amount of an anticancer agent incorporated into and/or onto thecollagen construct from about 1 day to about 60 days or any rangetherein, such as between about 1 day to about 30 days or between about 1day to about 20 days after implantation. In certain embodiments, about30% to about 40% of the total amount of an anticancer agent incorporatedinto and/or onto the collagen construct is released after about 30 daysafter implantation.

In other aspects, after about 30 days after implantation, about 30% toabout 100% or any range therein, such as about 30% to about 70%, orabout 35% to about 50% of the total amount of an anticancer agentincorporated into and/or onto the collagen construct can remainincorporated into and/or onto the collagen construct. In certainembodiments, about 30% to about 100% or any range therein, such as about30% to about 70%, or about 35% to about 50% of the total amount of ananticancer agent incorporated into and/or onto the collagen constructcan be released from about 30 days to about 100 days after implantation,about 30 days to about 3 years after implantation, or for the rest ofthe useful lifetime of the collagen construct.

In further aspects, the collagen construct can have a third and/orextended release of an anticancer agent from the collagen construct. Theintermediate release can provide for a prolonged release, in comparisonto the immediate and/or intermediate release described above, of theanticancer agent from the collagen construct. In some embodiments of theinvention, a portion and/or the remainder of an anticancer agent, e.g.,platinum, incorporated into and/or onto the collagen construct can bereleased or eluted from the collagen construct as it breaks down,absorbs, delaminates, denatures, etc. The amount of the anticancer agentreleased from the collagen construct as it breaks down, absorbs,delaminates, denatures, etc. can be from about 30% to about 100% or anyrange therein, such as about 30% to about 70%, or about 35% to about 50%of the total amount of an anticancer agent incorporated into and/or ontothe collagen construct.

The anticancer agent, e.g., platinum, can be released at a substantiallyconstant release rate from the collagen construct or at one or moredifferent release rates from the collagen construct over a period oftime. In particular embodiments, the anticancer agent is released at atleast three different rates over time. For example, about 15% to about35% of the total amount of an anticancer agent incorporated into and/oronto a collagen construct can be released or eluted from the collagenconstruct after about 1 day after implantation and about 50% to about70% of the total amount of an anticancer agent incorporated into and/oronto a collagen construct can be released after about 30 days afterimplantation. The remainder of the anticancer agent present in thecollagen construct or a portion thereof can be released for the rest ofthe lifetime of the collagen construct and/or as it breaks down,absorbs, delaminates, denatures, etc. at a constant rate, a variablerate, an intermittent rate, or any combination thereof. In particularembodiments, a release of an anticancer agent is a therapeuticallyeffective amount of the anticancer agent.

In certain aspects, the invention relates to a biocompatiblecross-linked collagen construct comprising a collagen construct that ismodified so as to bear multiple quinone and/or catechol groups, to whichanticancer agents, such as platinum, can bind with affinity. In theseaspects, the cross-linked collagen construct is in one respect anaffinity based carrier for which one may use commonly understoodtechniques relating to equilibrium constants to control the level ofanticancer agent that binds to the cross-linked collagen construct, inorder to effect particular desired release rates. In this regard, thebinding affinity of platinum to collagen constructs can be adjusted asneeded using several techniques. For example, the binding affinity canbe adjusted by modifying the amount of cross-linking agent (e.g. NDGA)that is reacted with the collagen. In another example, one or moreoxidizing and/or reducing agents can be used to modify and/or change theoxidation state of a functional group present in the cross-linkedcollagen construct, thereby altering the binding affinities of theanticancer agent for the cross-linked collagen construct. In thisregard, the anticancer binding affinities, referred to hereinabove ashaving at least three affinity phases, can be altered depending on thedesired rate of release. Accordingly, in certain aspects, a variety ofrelease rates can be achieved.

Particular embodiments of the present invention provide a method oftreating and/or preventing a disease and/or clinical symptom comprising:a) implanting a construct of the present invention in a subject, whereinthe construct comprises collagen and an anticancer agent incorporatedinto and/or onto the construct to provide a total amount of theanticancer agent in the construct, and b) releasing or delivering theanticancer agent in a plurality of in vivo releases. The collagen of theconstruct can comprise natural collagen and/or synthetic collagen in anyform. In some embodiments of the present invention, the collagencomprises at least one elongate synthetic collagen fiber, more typicallya plurality of elongate synthetic collagen fibers.

In particular embodiments, after implantation of the construct in asubject, the construct delivers a therapeutically effective amount ofthe anticancer agent in one or more in vivo releases of the anticanceragent from the construct. In other embodiments, the plurality of in vivoreleases, as measured in a saline solution at 37° C., comprise a firstrelease (R1) from t=0 to t=about 1 day after implantation and a secondrelease (R2) from t=about 1 day to t=about 30 days after implantation.In some embodiments of the present invention, the anticancer agent is ananticancer agent, such as platinum, that is used to treat and/or preventand/or inhibit cancer.

FIG. 1 is a flow chart of operations that can be used to carry outembodiments of the present invention. The one or more anticancer agentscan be incorporated into and/or onto the collagen constructs of thepresent invention at any time after a collagen construct (e.g., collagenfiber or a device with such fibers) is provided (block 500). In someembodiments of the present invention, the anticancer agent isincorporated into and/or onto a collagen fiber of the collagen constructbefore it is formed. In other embodiments, the anticancer agent isincorporated into and/or onto a collagen fiber of the collagen constructafter it is formed into a pre-final or final configuration/shape.

The anticancer agent can be incorporated into and/or onto a collagenconstruct during and/or after polymerization of a collagen fiber in theconstruct with a suitable cross-linker, such as, for example, NDGA(block 510). The amount of cross-linker present in the collagenconstruct can be less than about 15%, typically between about 10% toabout 1%, such as between about 5% to about 1%. In particularembodiments of the present invention, the amount of cross-linker, e.g.,NDGA, present in the collagen construct is less than about 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or any otherrange therein. Accordingly, the amount of collagen present in thecollagen construct can be more than about 85%, typically between about90% to about 100%, such as between about 90% to about 95%. In particularembodiments, the amount of collagen in the collagen construct is morethan about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or any other range therein. In some embodiments, theamount of polymerized collagen is increased to increase the amount of ananticancer agent incorporated into and/or onto a collagen construct. Thecollagen construct can further comprise non-collagenous components orbiocompatible materials, such as particulates, hydroxyapatite and othermineral phases, or drugs that facilitate tissue growth or other desiredeffects. See, U.S. Pat. No. 6,821,530, the contents of which areincorporated herein by reference above.

The cross-linking agent can be any suitable polymerizing (i.e.,cross-linking) material, such as, but not limited to, NDGA,3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxybenzaldehyde,3,4-dihydroxybenzoic acid, carbodiimide, glutaraldehyde, formaldehyde,tannic acid, isocyanates, and epoxy resins. In other embodiments, thecross-linking agent can be any suitable polymerizing material in whichat least one reactive group of the peptide is part of a diamino acid,such as but not limited to, lysine, arginine, asparagine, cysteine,glutamine, histidine and ornithine. In these aspects, hydroxyl groupsand mercapto groups on the peptide may contribute to the cross-linkingreaction. In other aspects, a dicarboxylic acid may be used as thecross-linking agent, thereby introducing a hydrocarbon bridge in-betweenthe cross-linked sections having a free amino, hydroxyl or thiol group.In particular embodiments, the cross-linking agent comprises a quinonegroup and/or a catechol group. Exemplary cross-linking agents that cancomprise a quinone and/or catechol functional group include, but are notlimited to NDGA, 3,4-dihydroxyphenylalanine, and dopamine. Thus, thepolymerized collagen can comprise one or more quinone and/or catecholgroups. In certain embodiments, the cross-linking agent is selectedbased on the anticancer agent(s) desired to be incorporated into and/oronto the collagen construct. For example, when platinum is desired as ananticancer agent, a cross-linking agent that comprises a quinone and/orcatechol functional group, e.g., NDGA, or that comprises one or morebasic nitrogen atoms, is selected.

Once an anticancer agent is provided (block 550), the collagen constructcan be contacted with an anticancer agent to incorporate the anticanceragent into and/or onto the collagen construct (block 580). The collagenconstruct can be contacted with an anticancer agent via a loadingsolution (i.e., a solution comprising the anticancer agent) for a periodof time sufficient to allow for the anticancer agent to be incorporatedinto and/or onto the construct. Alternatively and/or in addition tocontacting the collagen construct with a loading solution, the collagenconstruct can be contacted with a powder, such a dry powder comprisingan anticancer agent. The term “contacting” as used herein in referenceto the incorporation of the anticancer agent into and/or onto theconstruct, is intended to include treating, soaking, suspending,immersing, saturating, dipping, wetting, rinsing, washing, submerging,emersing, spraying, rolling, and/or any variation and/or combinationthereof. The construct can be contacted with an anticancer agent for aperiod of time of about 1 minute to about 24 hours or more. In someembodiments, the contacting step can be carried out for a period of timeof about 1 minute to about 36 hours or any range therein, such as about1 hour to about 24 hours or about 10 hours to about 20 hours. Afterbeing contacted with the one or more anticancer agents, the constructcan be washed with water and/or a suitable buffer. The buffer can be anybuffer, including, but not limited to, for example, sodium acetate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or3-(N-morpholino) propanesulfonic acid (MOPS) at a pH of about pH 6.5 toabout 7.8. The pH of the buffer can be about 6.5, 6.6, 6.7, 6.8, 6.9,7.0, 7.1, 7.2, 7.3, 7.5, 7.4, 7.6 or 7.8. The collagen construct canthen be dried.

The contacting step can be carried out at a temperature between about 5°C. to about 80° C. or any range therein, such as between about 10° C.and about 60° C., about 15° C. to about 40° C., or about 20° C. andabout 30° C. In particular embodiments, the temperature is about 25° C.The contacting step can be carried out at atmospheric pressure, reducedpressure (e.g., vacuumized pressure), high pressure, and/or anycombination thereof. In particular embodiments, the pressure isatmospheric pressure.

The amount of an anticancer agent incorporated into and/or onto thecollagen constructs of the present invention can be influenced byvarying different factors in the method of incorporating the anticanceragent. For instance, whether there are more than one anticancer agentsin the loading solution can affect the amount of an anticancer agentincorporated into the collagen construct. The concentration of the oneor more anticancer agents in the loading solution can also affect theamount of an anticancer agent incorporated into the collagen construct.The concentration of the one or more anticancer agent in the loadingsolution can range from about 0.1% to about 50%, such as about 0.5% toabout 35% or about 1% to about 20%. In particular embodiments, theconcentration of an anticancer agent in the loading solution is about0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, or any other range therein. In some embodiments of thepresent invention, the concentration of an anticancer agent in theloading solution is from about 0.1% to about 5%. In other embodiments,the loading solution can be a saturated or a supersaturated solution ofthe solute, i.e., the anticancer agent.

The pH of the loading solution as well as any additional components inthe loading solution can influence the amount of an anticancer agentthat is incorporated in the collagen construct. In some embodiments, thepH of the loading solution is between about pH 3-9, such as about pH 3,4, 5, 6, 7, 8, 9 or any other pH value therein. Additional components inthe loading solution can include, but are not limited to, agents thataid with the incorporation of the anticancer agent into and/or onto thecollagen construct, agents that modify the collagen construct such asoxidizing agents and reducing agents, agents that aid with thesolubility of the anticancer agent, agents that modify the pH of theloading solution, and any combination thereof. Further, other anticanceragents, non-collagenous components or biocompatible materials, such asparticulates, hydroxyapatite and other mineral phases, or drugs thatfacilitate tissue growth or other desired effects can be present in theloading solution. See, U.S. Pat. No. 6,821,530,

In certain embodiments of the present invention, the collagen constructis contacted with an agent that modifies the collagen construct (block520). The agent can aid in incorporating an anticancer agent into and/oronto the collagen construct by increasing the amount of the anticanceragent bound and/or the enhancing and/or strengthening chemical bondbetween the anticancer agent and the collagen construct. The modifyingagent can alter a functional group present in the collagen construct toincrease and/or enhance the incorporation of an anticancer agent intoand/or onto the collagen construct. In some embodiments, the modifyingagent can change the oxidation state of a functional group present inthe collagen construct. In these embodiments, reducing and/or oxidizingagents may be used to by one of ordinary skill in the art to modifyand/or change the binding affinity of the anticancer agent for thecollagen construct. In particular embodiments, a functional grouppresent in the collagen construct is modified to be a quinone group or acatechol group. The modifying agent, in other embodiments, blocks (e.g.,partially or completely) a functional group present in the collagenconstruct. Blocking, and optionally later unblocking a functional group,can allow for one or more anticancer agents and/or other materials to beincorporated into and/or onto the collagen construct.

The method of incorporating an anticancer agent into the collagenconstructs of the present invention can be repeated to add more of thesame anticancer agent and/or to add one or more additional anticanceragents.

II. Exemplary Collagen Construct

The collagen constructs of the present invention comprise collagen,typically dermal collagen. However, the collagen can be of any form andfrom any origin. The collagen can be any of the identified collagengenotypes, for example, the interstitial fiber forming collagen types I,II and III, as well as any other substantially fiber forming types ofcollagen, for example collagen VI. The collagen can be acid solublecollagen or pepsin solubilized or soluble collagen. The collagen can befrom mammalian cells synthesized in vitro. The collagen can be frommolecularly engineered constructs and synthesized by bacterial, yeast orany other molecularly manipulated cell type. For example, the collagencan be sea cucumber dermis collagen, bovine, caprine, porcine, ovine orother suitable donor mammal, marine animal collagen such as chinoderms,molecularly engineered collagen, or gelatin (e.g., in any suitable formincluding solid, gel, hydrogels, liquids, or foams). In addition, thecollagen can be digested with a protease before, where used, oxidizingand polymerizing steps. The collagen can be in the form of microfibrils,fibrils, natural fibers, or synthetic fibers.

In some embodiments, the collagen can be solubilized, dissolved orotherwise transferred into an acid solution, for example, acetic acid(e.g., about 0.01 M to about 1.0 M, typically about 0.5 M), hydrochloricacid (between about pH 1 to about pH 3, typically about pH 2.0), or anyother suitable acid at appropriate concentration (e.g., about pH 1.0 toabout pH 3.0, typically about pH 2.0). Dialysis may optionally be usedto neutralize a soluble collagen solution. The collagen can also oralternatively be dissolved in a neutral buffered solution either with orwithout salts, e.g., phosphate buffer at about pH 7.0, or phosphatebuffered saline at about pH 7.0. The phosphate buffer can be at anyconcentration of sodium phosphate between about 0.01 M and about 0.5 M,but more typically between about 0.02 M and about 0.1M. The buffer canalso be any buffer, including, but not limited to, for example, sodiumacetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or3-(N-morpholino) propanesulfonic acid (MOPS). The collagen can bepresent in a quantity that is at least about 0.1% to about 10%,typically between about 0.1% to about 5% (e.g., about 0.1, 0.2, 0.3,0.4, 1.0, 2.0, 4.0%) weight per volume, or weight per volume in theneutral buffer solution before fibrillogenesis and fiber formation. In adried fiber collagen, collagen can be present in an amount of weight byvolume of between about 50-100% (e.g., at least about 75%, 90%, 95% or100%) before crosslinking (where crosslinking is used).

Collagen “microfibrils,” “fibrils,” “fibers,” and “natural fibers” referto naturally-occurring structures found in a tendon. Microfibrils areabout 3.5 nm to about 50 nm in diameter. Fibrils are about 50 nm toabout 50 μm in diameter. Natural fibers are above about 50 μm indiameter. A “synthetic fiber” refers to any fiber-like material that hasbeen formed and/or chemically or physically created or altered from itsnaturally-occurring state. For example, an extruded fiber of fibrilsformed from a digested tendon is a synthetic fiber but a tendon fibernewly harvested from a mammal is a natural fiber.

Of course, synthetic collagen fibers can include non-collagenouscomponents or biocompatible materials, such as particulates,hydroxyapatite and other mineral phases, or drugs that facilitate tissuegrowth or other desired effects. See, U.S. Pat. No. 6,821,530,incorporated herein by reference above. For example, the fibers and/orconstructs formed from same, can include compositions that can containcarbon nano-tubes, zinc nano-wires, nano-crystalline diamond, or othernano-scale particulates; and larger crystalline and non-crystallineparticulates such as calcium phosphate, calcium sulfate, apatiteminerals. For example, the compositions can also or alternativelycontain anticancer agents such as bisphosphonates, anti-inflammatorysteroids, growth factors such as basic fibroblast growth factor, tumorgrowth factor beta, bone morphogenic proteins, platelet-derived growthfactor, and insulin-like growth factors; chemotactic factors suchfibronectin and hyaluronan; and extracellular matrix molecules such asaggrecan, biglycan, decorin, fibromodulin, COMP, elastin, and fibrillin.In some embodiments, the fibers and/or fiber-derived constructs cancontain cells, engineered cells, stem cells, and the like. Combinationsof the above or other materials can be embedded, coated and/or otherwisedirectly or indirectly attached to the collagen fibers and/or constructformed of same.

The term “collagen gel” means a semi-solid (e.g., gelatinous density)material that includes collagen fiber, fibrils and/or microfibrils,typically dermal collagen, that has been acid or pepsin solubilized(e.g., soluble collagen) and processed to maintain the collagen in itsmolecular form. The collagen concentration of the soluble collagenand/or resulting soluble collagen gel can be between about 0.1% to about4% weight per volume. The collagen can be solubilized, dissolved, and/orsuspended in a solution (e.g., water or buffer solution). The solutioncan be a neutralized solution with a pH of about pH 7.0 to about 7.4.The pH can be about 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments thepH is about 7.2. The buffer can be any buffer, including, but notlimited to, for example, sodium acetate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or3-(N-morpholino) propanesulfonic acid (MOPS) at a pH of about pH 7.0 toabout 7.4. The soluble collagen gel may be formed to be in a cylindricalshape of a defined length and diameter, typically with a diameter ofbetween about 0.1 cm to about 1 cm, and a length of between about 5 cmto about 100 m, more typically between about 1 m to about 50 m.

The collagen gel can comprise non-collagenous components orbiocompatible materials, such as one or more particulates and/orminerals. Exemplary minerals include, but are not limited to, calciumphosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate,monotite, brushite, calcium pyrophosphate, tricalcium phosphate,tetracalcium phosphate, octacalcium phosphate, amorphous calciumphosphate, hydroxyapatite, carbonateapatite, calcite, and calciumsulfate. One or more minerals can be present in a quantity from about0.1% to about 5%, typically between about 0.1% to about 1% (e.g., 0.1,0.2, 0.4, 0.6, 0.8, or 1%) weight per volume. When one or more mineralsand/or particulates are present in the collagen gel, the collagen gelcan be used to create a rough or textured surface. “Rough” as usedherein refers to an unequal or varied surface that can contain surfacetexture, ridges, and/or bumps. In some embodiments at least one mineralis present in the collagen gel to create a rough inner and/or outersurface. The higher the mineral concentration in the collagen gel,typically, the rougher the surface and/or resulting tube. A high mineralconcentration can provide a surface and/or a tube that is lighter incolor than a surface and/or tube containing no minerals.

The collagen fibers and collagen gel can be produced in batch orcontinuous-type systems, including wet gel collagen extrusion systems,which produce cylindrical lengths of gel that can be allowed tosubstantially dry (actively or passively) to obtain a suitable length offiber. Examples of some collagen fiber production processes that cangenerate soluble collagen in suitable lengths are described in U.S. Pat.No. 6,565,960, and pending U.S. Patent Application Publication No.US-2008-0188933-A1, the contents of which are hereby incorporated byreference.

The collagen fibers can be spooled for supplying to an automated orsemi-automated winder to form the biomedical construct. The collagenfibers may be formed with a relatively thin diameter, such as, forexample between about 0.05 mm to about 0.2 mm (average), such as about0.08 mm dry diameter (average) and about a 0.13 mm wet diameter(average). A collagen fiber can be an elongate continuous length offiber formed of denatured (gelatin) and/or non-denatured collagen (e.g.,whole or fragmented native collagen fibers from tendon, skin, or othersources). The fiber can have a length of at least about 0.25 inches,typically greater than about 0.5 inches, such as between about 1-30inches or between about 1 m to about 100 m. In certain embodiments ofthe present invention, a plurality of elongate collagen fibers can beutilized. In particular embodiments of the present invention, anelongate collagen fiber is polymerized before and/or after it is used toprepare a construct.

The term “gelatin” refers to denatured collagen. Gelatin can be derivedfrom collagen in a well known manner or can be obtained from commercialsuppliers, such as Sigma-Aldrich®, located in St. Louis, Mo. Anexemplary method of obtaining gelatin is by heating collagen at asuitable temperature to cause it to become denatured. Denaturationresults in the irreversible transformation of collagen into a randomcoiled structure, which is gelatin. Gelatin can be derived from one ormore sources of collagen and derived from one or more types of collagen,such as but not limited to, types I, II, III, and/or VI. Exemplarysources from which gelatin is derived include, but are not limited to,sea cucumber dermis collagen, bovine, caprine, porcine, ovine or othersuitable donor mammal collagen, and marine animal collagen such aschinoderms. The gelatin can be derived from collagen obtained frommammalian cells synthesized in vitro. The gelatin can be derived fromcollagen obtained from molecularly engineered constructs and synthesizedby bacterial, yeast or any other molecularly manipulated cell type.

The term “gelatin slurry” as used herein refers to a mixture of gelatinin a solvent (e.g., water or buffer solution). The gelatin slurry can bea homogeneous or heterogeneous mixture. Gelatin in the gelatin slurrycan be suspended, solubilized, and/or dissolved (e.g., completely orpartially) in a solvent to form a gelatin slurry. The gelatin slurry cancomprise other components, such as, but not limited to, one or moreminerals and/or particulates, that can be suspended, solubilized, and/ordissolved in the solvent. The buffer can be any buffer, including, butnot limited to, for example, sodium acetate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or3-(N-morpholino) propanesulfonic acid (MOPS) at a pH of about pH 6.5 toabout 7.8. The pH of the buffer can be about 6.5, 6.6, 6.7, 6.8, 6.9,7.0, 7.1, 7.2, 7.3, 7.5, 7.4, 7.6 or 7.8. In some embodiments the pH isabout 7.2. The gelatin can also or alternatively be dissolved in aneutral buffered solution either with or without salts, e.g., phosphatebuffer at about pH 6.5 to about 7.8, or phosphate buffered saline atabout pH 6.5 to about 7.8. The phosphate buffer can be at anyconcentration of sodium phosphate between about 0.01 M and about 0.5 M,but more typically between about 0.02 M and about 0.1 M. The gelatin canbe present in a quantity from about 0.1% to about 60%, typically betweenabout 2% to about 40% (e.g., about 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25,30, 35, or 40%) weight per volume.

The gelatin slurry can be heated to create a viscous slurry at atemperature that keeps the gelatin from gelling or solidifying duringapplication, and/or to dissolve or solubilize the gelatin in thesolvent. When a gelatin slurry is cooled to a sufficient temperature a“gelatin hydrogel” is formed. The term “gelatin hydrogel” as used hereinrefers to a semi-solid (e.g., gelatinous density) material formed by thegelatin slurry that includes gelatin and can comprise other components,such as, but not limited to, one or more minerals and/or particulates.The gelatin in the gelatin slurry and in the resulting gelatin hydrogelare composed of denatured collagen and cannot be used to producecollagen fibers, fibrils, and/or microfibrils. To be clear, in contrast,the term “collagen gel” as used herein refers to a gel that includescollagen fiber, fibrils and/or microfibrils that has been acid or pepsinsolubilized (e.g., soluble collagen) and processed to maintain thecollagen in its molecular form, whereas the terms “gelatin hydrogel” and“gelatin slurry” as used herein refer to compositions of gelatin, whichis denatured collagen that cannot be used to produce collagen fibers,fibrils, and/or microfibrils. Stated differently, gelatin is denaturedcollagen which does not maintain collagen in its molecular form since itis irreversibly transformed into a random coiled structure.

The gelatin slurry and/or the gelatin hydrogel, which may or may not beattached to at least one collagen fiber, can be cross-linked with asuitable polymerizing (i.e., cross-linking) material, such as, but notlimited to, NDGA, 3,4-dihydroxyphenylalanine, dopamine,3,4-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, carbodiimide,glutaraldehyde, formaldehyde, tannic acid, isocyanates, and epoxyresins, or may be used in a non-cross-linked state. In particularembodiments, the cross-linker comprises a quinone group and/or catecholgroup. Exemplary cross-linking agents that can comprise a quinone and/orcatechol functional group include, but are not limited to NDGA,3,4-dihydroxyphenylalanine, and dopamine. Thus, the polymerized collagencan comprise one or more quinone and/or catechol groups.

Alternatively or in addition, the gelatin slurry and/or gelatin hydrogelcan be stabilized with treatments, such as, but not limited to, one ormore of dehydrothermal treatment, glycation, and ultraviolet light. Thegelatin slurry and/or the gelatin hydrogel treated with a polymerizingmaterial and/or a stabilization treatment can be resistant toliquification at 37° C. and/or thermally stable at temperatures overabout 37° C. The gelatin slurry and/or the gelatin hydrogel treated witha polymerizing material and/or a stabilization treatment can bethermally stable at temperatures up to about 120° C., typically attemperatures between about 37° C. to about 104° C. The polymerizedand/or stabilized gelatin hydrogel can be stronger and/or stiffer thanan untreated gelatin slurry and/or gelatin hydrogel (e.g., an untreatedgelatin hydrogel has a compressive stiffness of about 0.70 MPa, comparedto about 4.71 MPa for NDGA-treated gelatin hydrogel). The polymerizedand/or stabilized gelatin hydrogel can be nearly elastic under dynamiccompression loads (e.g., rebounds substantially completely aftercompression to over 80%, while untreated gelatin hydrogels fracture whencompressed to 80%). The polymerized and/or stabilized gelatin hydrogelcan undergo large deformations without comprising its mechanicalproperties. According to some embodiments, the gelatin slurry and/or thegelatin hydrogel, if polymerized (i.e., cross-linked) and/or stabilized,can be polymerized and/or stabilized at any time either before, during,and/or after application and/or drying to at least one collagen fiber,where applied.

The gelatin slurry can be heated prior to application typically aboveroom temperature, such as up to about 120° C. or even more. In someembodiments, the gelatin slurry can be heated and/or kept at betweenabout room temperature and about 100° C., typically between about roomtemperature and about 70° C. to keep the gelatin from gelling orsolidifying during application, and/or to dissolve or solubilize thegelatin and/or one or more minerals in the solvent.

During application of the gelatin slurry onto a construct (e.g.,collagen fiber), the gelatin slurry can be heated above roomtemperature, such as between about 20° C. and about 70° C., betweenabout 20° C. and about 60° C., typically between about 45° C. to about55° C. to keep the gelatin from gelling or solidifying duringapplication, and/or to dissolve or solubilize the gelatin and/or one ormore minerals in the solvent.

The gelatin slurry can be heated by known methods and devices, such as,but not limited to, heating using a water bath, heating block, heatingpad, solar or light source, microwave, or bunsen burner. The temperatureto which the gelatin slurry is heated can depend on the concentration ofgelatin and/or other components present in the slurry. Typically, if ahigh concentration of gelatin and/or other components is present in thegelatin slurry, then the gelatin slurry may need to be heated to ahigher temperature to create a viscous slurry at a temperature thatkeeps the gelatin from gelling or solidifying during application, and/orto dissolve or solubilize the gelatin and/or other components in thesolvent. Generally, the higher the concentration of gelatin in theslurry, the higher the temperature needed to create a viscous slurry ata temperature that keeps the gelatin from gelling or solidifying duringapplication, and/or to dissolve or solubilize the gelatin in thesolvent. However, other components present in the gelatin slurry, e.g.,minerals, may affect the viscosity of the gelatin slurry, thetemperature at which the gelatin slurry gels or solidifies, and/or thesolubility of the gelatin and/or minerals in the solvent. Thus, thetemperature to which the gelatin slurry is exposed or heated to canvary.

The term “film” refers to a thin layer of collagen gel, gelatin slurry(typically comprising one or more minerals), and/or gelatin hydrogel(typically comprising one or more minerals) that has dried. The collagengel, gelatin slurry, and/or gelatin hydrogel can be actively and/orpassively dried. Exemplary methods of drying the collagen gel, gelatinslurry, and/or gelatin hydrogel include, but are not limited to, airdrying, drying under heat, or drying in an oven or dryer usingconduction, convection, infrared/radiant, or radio frequency. Themoisture content of the resulting collagen film and/or gelatin film canbe less than about 25% by weight of the film, less than about 15% byweight of the film, but is typically less than about 5% by weight of thefilm to provide a state of the collagen film and/or gelatin film at alow moisture content.

Several layers of the collagen gel, gelatin slurry, and/or gelatinhydrogel can be applied or used to generate the desired film thicknessor coverage. For example, between about 1-20 layers of collagen gel,gelatin slurry, or gelatin hydrogel can be applied to form a collagenfilm or gelatin film, typically between about 1-10 layers of collagengel, gelatin slurry, or gelatin hydrogel can be applied to form acollagen film or gelatin film. In particular embodiments, between about1-20 layers of collagen gel are placed about an outer surface of asupport member and allowed to dry, then, at least one collagen fiber iswound a number of revolutions about a length of the support member andwhile winding the at least one collagen fiber between about 1-20 layersof a gelatin slurry are applied to the at least one collagen fiber andoptionally allowed to dry, then, between about 1-20 layers of collagengel are placed onto the at least one collagen fiber with the gelatinhydrogel or gelatin film and allowed to dry.

The one or more layers of collagen gel, gelatin slurry, and/or gelatinhydrogel can comprise different components and/or comprise the samecomponents present in different concentrations. In certain embodiments,each of the layers comprise the same components, e.g., minerals andparticulates, and in other embodiments the layers comprise differentcomponents. In particular embodiments, each of the layers comprise thesame components, but in each layer the concentration of the componentsis different. For example, in certain embodiments, the mineralconcentration in a first (inner) layer can be less than the mineralconcentration in the outer layer. The film can be present in a thicknessthat is between about 5 microns and about 1 mm, typically between about5 microns and about 700 microns, and more typically between about 5microns and about 500 microns. The film of gelatin hydrogel is typicallythicker than the film of collagen gel.

In some embodiments, a collagen gel, where used, can provide a smooth(and typically a substantially constant diameter) surface over and/orunder the at least one collagen fiber. In other embodiments, a collagengel comprising one or more minerals, e.g., hydroxyapatite, where used,can provide a rough layer (e.g., inner and/or outer surface) over and/orunder the at least one collagen fiber.

Methods of Treatment

Spectroscopic and chromatographic methods are employed to determine thesolution stability of the constructs provided herein. The complexes arescreened against, e.g., the L1210 mouse tumor model to determineantitumor activity. In addition the M5076 ovarian, H460 lung, B16melanoma, and Lewis lung murine tumor models may be used, e.g., asxenografts.

When used as antitumor agents, the platinum complexes of this inventioncan be administered in a manner and with a protocol comparable tocisplatin, carbopatin, oxaliplatin, and/or satraplatin. Theyadvantageously are administered to patients, humans or animals, havingtumors susceptible to therapeutic treatment by platinum complexes, assterile aqueous solutions. The solutions are preferably administeredintraveneously or intraarterially, although other forms ofadministration may be indicated in certain cases. Solutions forintravenous injections will normally be sterile physiological solutions,which may also contain appropriate amounts-of alkali, sodiumbicarbonate, to convert complexes bearing acidic water-solubilizinggroups to their salts. Suitable dosage forms can also include oily oraqueous injectable preparations, for intramuscular or intraperitonealinjection, and solid dosage forms, such as solid implants, capsules, andthe like.

The effective amounts of the complex of the invention which should beadministered can be determined by conventional methods which will beapparent to the skilled artisan. Normally, the activity of the platinumcomplex of this invention will be evaluated in a screen along with aknown complex such as ciplatin, carboplatin, oxaliplatin, and/orsatraplatin. The relative potency and the therapeutic index, the ratioof therapeutic effectiveness to toxicity, compared to that of the knownanalogue will normally determine the relative dosage compared toconventional dosages of the analogue for the type of cancer beingtreated.

The treatment regimen can be varied in ways which are well known to theskilled artisan, as a function of the type of cancer being treated, thecondition of the subject, and the particular properties of the antitumorplatinum complex being administered. Inevitably, a certain amount ofexperimentation is required to determine the optimum dosages andtreatment regimens, as is normally the case for anticancer therapy.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

In the following examples certain collagen fiber constructs areutilized. Such constructs (e.g., sleeves or tubes) are cross-linked withnor-dihydroguaiaretic acid (NDGA). However, this cross-linking agent isonly illustrative. The present invention is not intended to be limitedto cross-linked constructs where NDGA is the cross-linking agent. Forexample, other cross-linking agents, such as, but not limited to3,4-dihydroxyphenylalanine, dopamine, 3,4-dihydroxybenzaldehyde,3,4-dihydroxybenzoic acid, a carbodiimide, glutaraldehyde or other di-and multi aldehydes, formaldehyde, tannic acid, isocyanates such as di-and multi-isocyanates, di and multi-diazopyruvates, pluronics, and epoxyresins, and/or stabilization treatments, such as, but not limited to,one or more of dehydrothermal treatment, glycation, and ultravioletlight may be used in the present invention. In particular embodiments,the cross-linking agent comprises a quinone group and/or catechol group.Thus, the polymerized collagen can comprise one or more quinone and/orcatechol groups.

Example 1

FIG. 2 illustrates exemplary sleeves or tubes (10) of woundNDGA-collagen fibers that may be particularly suitable for medicalconstructs. The NDGA-collagen tubes comprise a solid sheet of type Icollagen cross-linked with NDGA (nor-dihydroguaiaretic acid) and can beprepared as described below in the materials and methods. The materialcan comprise greater than about 95% collagen and less than about 5% NDGApolymer. The diameter, length, and wall thickness of the collagenconstruct is scalable (FIG. 2). For instance, the inner diameter of thetube can vary between about 1 and 10 mm. The thickness of the wall canvary between about 0.1 and 3 mm. The length of the tube can vary, e.g.,from between about 1 to 6 cm or more.

The mechanical properties of the NDGA-collagen constructs are governedby fiber angle, wall thickness (wall thickness/diameter) and cross-linkdensity; all of which can be tuned to satisfy the mechanicalrequirements for the specific surgical application. NDGA-collagen isbiocompatible and biodegradable. Biologically active compounds can beincorporated into the biomaterial, including hydroxyapatite and TCPminerals, extracellular matrix macromolecules (glycosaminoglycans),anticancer agents such as platinum and bisphosphonates,anti-inflammatory drugs, and antibiotics.

It in contemplated that platinum can be incorporated into NDGA-collagenconstructs and that the platinum in the NDGA-collagen constructs canslowly elute from the construct in normal saline. Without being bound toany particular theory, platinum is believed to bind to and/or complexwith the NDGA-collagen due to the high concentration ofcatechols/quinones and catechol/quinone derivatives in the cross-linkedcollagen material. NDGA contains two catechols which, without beingbound to any particular theory, are contemplated to be responsible forcross-linking the collagen material through the generation of quinonesand covalent adduct formation. Quinones have been demonstrated to bindtransition and heavy metals. Biological materials that containconcentrated catechols/quinones can exhibit extraordinarily highaffinity for metals and in some cases can be considered metal chelatingcompounds (e.g., siderophores).

Materials and Methods

NDGA-collagen tubes (10) 8×25 mm are manufactured as described in U.S.pending U.S. Patent Application Publication No. 2010/0094318, which isincorporated herein by reference, using purified type I bovine collagen.The tubes (10) are within engineering specifications of the product(weight and dimensions—weight: about 100 mg, dimensions: about 8 mm indiameter and about 25 mm long). The dried tubes (10) are incubated in 1%(w/v) platinum (II) diaqua complex in water at room temperature for 16hours. The tubes are then washed with deionized water and dried.Platinum content in the NDGA-collagen is measured e.g., by InductivelyCoupled Plasma-Mass Spectrometry (ICP-MS). Results are expressed asμg/g, which is equivalent to parts per million (ppm).

Strength of Binding

The strength of platinum binding in NDGA-collagen material comprisingplatinum, is evaluated by washing the material in increasingconcentrations of a platinum chelator such as ammonium hydroxide or analkylene diamine such as ethylene diamine. The samples are washedthoroughly with water, then incubated at the indicated platinum chelatorconcentrations. The amount of platinum remaining in the material ismeasured.

Platinum Elution in Normal Saline

NDGA-collagen tubes comprising platinum are incubated in normal salineat 37° C. for 1, 3, 6, 13 and 30 days. At each time point, six samplesare collected, dried and the amount of platinum remaining in thematerial is measured.

Example 2 Platinum Binding in NDGA Cross-Linked Collagen Materials

To assess platinum binding in other NDGA cross-linked collagenousmaterials, samples of type I collagen fiber, bovine pericardium, porcinesmall intestinal submucosa (SIS), bovine tendon collagen sponge, bovinecollagen casing, and plain gut suture are treated with NDGA according toestablished protocols, such as those described in U.S. Pat. No.6,565,960, Koob, T. J. and Hernandez, D. H. “Material properties of NDGApolymerized collagen fibers: Development of biologically-based tendonconstructs” Biomaterials, 23, 203-212 (2002), and Koob, T. J. andHernandez, D. H. “Mechanical and thermal properties of novel polymerizedNDGA-gelatin hydrogels” Biomaterials 24, 1285-1292 (2002), which areincorporated herein by reference. The NDGA treated materials areincubated in 1% (w/v) platinum (+2) diaqua complex as described inExample 1, washed with de-ionized water, and dried. Platinum content inthe collagenous materials is measured on six replicate samples from eachcollagenous material. It is contemplated that the methods used toincorporate platinum into and/or onto the collagenous materials can beused on essentially any collagen based biomaterial.

The incorporation of platinum in collagenous materials can produce abiomaterial imbued with anticancer capabilities. As such, the method ofincorporating platinum into collagenous materials can provide a drugdelivery device, preferably for anticancer drug delivery.

Example 3 Weight Measurement and Metal Analysis of Exemplary Constructs

To determine the amount of platinum that can bind to gut sutures ofvarious lengths and thicknesses, weight measurements and metal analysisare conducted as follows:

Materials

The following specimens are evaluated as follows:

-   -   a. Size 2-0 Plain Gut Sutures    -   b. Size 2-0 Plain Gut Sutures doped with platinum    -   c. Size 2-0 NDGA-crosslinked 24 hrs Gut Sutures    -   d. Size 2-0 NDGA-crosslinked 24 hrs Gut Sutures doped with        platinum    -   e. 16-fiber braided NDGA-crosslinked collagen yarn (15        picks/inch, manufactured by Steeger USA, Inman, S.C.).

Each specimen evaluated is 60 mm long, and two specimens are evaluatedfor each group.

Metal Analysis

Three groups of specimens are prepared as follows:

Group 1: 2-0 plain gut+platinum

Group 2: 2-0 NDGA gut+platinum

Group 3: 16-fiber yarn suture+platinum

Furthermore, five specimens from each of the groups are evaluated, forwhich each specimen is 20 mm in length. The weight of each specimen ismeasured in CE using a 4-digit balance, and metal analyses are conductedpursuant to standard protocols.

Example 4 Stability Studies

The stability of the crosslinked collagen-Pt constructs are determinedby measuring the ultraviolet spectra of the constructs in solution, run,e.g., at half-hour intervals. The decrease in absorbance with time is ameasure of complex decomposition.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although certain illustrative embodimentsof this invention have been described, those skilled in the art willreadily appreciate that many modifications of these are possible withoutmaterially departing from the novel teachings and advantages of thisinvention. Accordingly, all such modifications are intended to beincluded within the scope of this invention as defined in the claims.The invention is defined by the following claims, with equivalents ofthe claims to be included therein.

The invention claimed is:
 1. A method of manufacturing a therapeuticallyeffective anticancer cross-linked collagen construct comprisingcross-linked collagen and an amount of a releasable platinum-containinganticancer agent, wherein said cross-linked collagen comprises collagenand one or more cross-linking agents which cross-link the collagen insaid construct, wherein the platinum is incorporated into the constructby chelation to said agents, and wherein the platinum is capable ofbeing released in vivo when administered to a subject, the methodcomprising contacting the cross-linked collagen with a cis-diaqua or acis-dihalo platinum complex to provide the therapeutically effectiveanticancer cross-linked collagen construct.
 2. A method of treating asubject suffering from cancer amenable to treatment with platinumalkylators, the method comprising administering a therapeuticallyeffective amount of an anticancer cross-linked collagen construct to thesubject, wherein the construct comprises (a) cross-linked collagencomprising collagen and one or more cross-linking agents which crosslink the collagen in said construct and (b) an anticancer amount of areleasable platinum-containing anticancer agent incorporated into theconstruct, wherein at least a portion of the platinum incorporated intothe construct is incorporated by chelation to said agents, and whereinthe platinum is capable of being released in vivo when administered tothe subject.
 3. The method of claim 2, wherein the anticancercross-linked collagen construct provides plurality of in vivo releaserates.
 4. The method of claim 2 wherein the anticancer cross-linkedcollagen construct provides a sustained release of platinum, wherein thesustained release comprises a plurality of release rates comprising animmediate release, an intermediate release, an extended release or anycombination of release rates thereof.
 5. The method of claim 1 whereinat least one of the cross-linking agents comprises a 1,2-benzoquinoneand/or a 1,2-dihydroxy phenyl moiety.
 6. The method of claim 1 where theplatinum is chelated at least in part to the benzoquinone and/or the1,2-dihydroxyphenyl moiety.
 7. The method of claim 2 wherein at leastone of the cross-linking agents comprises a 1,2-benzoquinone and/or a1,2-dihydroxy phenyl moiety.
 8. The method of claim 2 where the platinumis chelated at least in part to the benzoquinone and/or1,2-dihydroxyphenyl moiety.